Method to recover bioactive compounds

ABSTRACT

The invention relates to a process for separating bioactive compounds obtained from vegetable materials. The invention also relates to a process for extracting bioactive compounds from vegetable material.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of commonly assigned copendingU.S. patent application Ser. No. 12/682,394, filed on Jul. 9, 2010,which is the National Stage of International Application No.PCT/AU2008/001495, filed on Oct. 9, 2008, which claims the priority dateof Australian Application No. 2007905554, filed on Oct. 10, 2007. Thecontents of each prior application are hereby incorporated by referencein their entirety.

BACKGROUND

1. Field of the Invention

The present invention relates to a process for separating bioactivecompounds obtained from vegetable material. The present invention alsorelates to a process for extracting bioactive compounds from vegetablematerial.

2. Background Information

is Plants and vegetable matter contain a range of compounds which arebiologically active in humans providing beneficial physiologicaleffects, including a reduction in the risks of cancer, heart disease andarthritis.

A range of bioactive compounds can be found in a wide variety of plantand vegetable material. Citrus fruits for example, contain bioactivecompounds that can be included in two major groups; the limonoids andthe flavonoids.

The limonoids are triterpenoid compounds which usually occur in citrusfruits. The limonoids may exist as aglycones, or be linked to a glucosemolecule (the glucoside). The limonoid glucosides have recently beenshown to possess powerful anti-cancer properties in animals.

The flavonoids are a group of benzopyran derivatives which occur widelyin plants. The flavonoids typically consist of a benzene ring fused witha heterocyclic six-membered ring containing an oxygen atom. Manyflavonoids may also exist as glycosides. In citrus fruits, the mostpredominant flavonoids are the flavanones, narirutin and hesperidin (inorange) and naringin (in grapefruit). These compounds are capable oflowering blood cholesterol levels in hypercholesterolemic individuals.

The flavonoids in citrus also include the polymethoxylated flavones.This group of compounds is represented by flavones substituted bymethoxy groups and is unique to citrus. The polymethoxylated flavoneshave a wide range of physiological effects, including a very highantioxidant capacity, which has prompted investigations into theirpotential use as a potent anti-cancer agent and as an anti-inflammatoryagent.

Polyphenolic compounds such as the citrus limonoids and citrusflavonoids occur in significantly higher concentrations in peel tissuewhen compared to the concentration in endocarp from which juice isextracted. The high concentrations of these compounds in the peel tissuehelp form the basis of the plant's protective mechanisms againstbacteria, moulds, yeasts and insects.

Citrus peel is bitter, most often because of the presence of limonoidcompounds in their aglycone form, and peel discharged from juicingoperations is usually limed, pressed, dehydrated, pelletised and used asstock feed.

Recently, a commercial practice has arisen in the citrus processingindustry to extract water soluble compounds from the peel of citrusfruits using a range of devices. The resultant dilute water extract(“juice”) is bitter and after clarification (or partial clarification)this juice is de-bittered by passing it over a synthetic polymeradsorbent. In this way, the bitter principles which are adsorbed to thepolymer can be separated from the natural sugars and acids and someflavour compounds, which are not adsorbed to the polymer. Most limonoidsand flavonoids however may also be preferentially adsorbed by thepolymer along with the bitter principles. Treatment of the polymer witha caustic soda solution desorbs these compounds to regenerate thepolymer. The treatment however also destroys the bioactive compounds,which are discharged as waste with the spent caustic soda solution.

One process for extracting bioactive components from citrus fruits hasbeen described in PCT/AU01/01113 (WO 02/20112). In this process, a rawcitrus extract is passed over a polystyrene-divinyl benzene polymer andbioactive compounds from the raw material are adsorbed onto the polymer.The bioactives are then sequentially eluted from the de-bitteringpolymer adsorbent by a constant gradient concentration of alcohol in analcohol water mixture. Three separate alcoholic extracts containinglimonoid glucosides, flavanone glycosides and polymethoxylated flavonesare then able to be collected from the polymer adsorbent.

While this process enables the valuable bioactive compounds to berecovered from the adsorbent polymer, some mixing of the bioactivecompounds in the eluent fractions can occur, leading to incompleteseparation of the different bioactives from each other, in particular ofthe limonoid glucosides from the flavanone glycosides. This may resultin a lower purity leading to formulation difficulties, for example.

It would be desirable to address some or all of these problems and toprovide an improved process for obtaining bioactive compounds fromvegetable material such as citrus fruits.

SUMMARY

The present invention relates in one aspect to a process for theselective separation of bioactive compounds, the process comprising thesteps of:

(a) contacting a plurality of bioactive compounds with a polymeradsorbent under conditions allowing adsorption of at least one bioactivecompound on to the adsorbent while at least one bioactive compound isnot adsorbed on to the adsorbent; and

(b) collecting a solution comprising at least one bioactive compoundwhich has not adsorbed onto the adsorbent.

The present invention relates in another aspect to a process forpurifying a bioactive compound comprising the step of contacting thebioactive compound with an ion exchange resin under conditions allowingionic interactions between the bioactive compound and the resin suchthat the bioactive compound is adsorbed on to the resin.

The present invention also relates in a further aspect to a process forthe selective extraction of bioactive compounds from a vegetablematerial, the process comprising the step of contacting the vegetablematerial with a solvent under conditions to extract at least one watersoluble bioactive compound from the vegetable material to therebyprovide an extract comprising the water soluble bioactive compound and avegetable residue comprising at least one water insoluble bioactivecompound.

Yet a further aspect of the present invention provides a bioactivecompound produced by a process as described herein.

A further aspect of the invention provides a composition comprising alimonoid glycoside present at a purity of greater than about 10%, 50%,or 70%.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention description below refers to the accompanying drawings, ofwhich:

FIG. 1 shows a HPLC chromatogram illustrating the components present ina representative raw extract obtained from orange peel.

FIG. 2 shows a HPLC chromatogram illustrating the presence of sugars andorganic acid compounds in a fraction of an eluate collected after theorange peel extract is passed over an acrylic polymer adsorbent inaccordance with one embodiment of the invention.

FIG. 3 shows a HPLC chromatogram illustrating the presence of limoninglucoside (LG) in a fraction of collected eluate.

FIG. 4 shows a HPLC chromatogram illustrating the presence of limoninglucoside, a related limonoid, nomilin glucoside and obacunone glucosidein a fraction of collected eluate.

FIG. 5 shows a HPLC chromatogram illustrating the presence of flavonoidshesperidin and narirutin in a fraction of collected eluate.

FIG. 6 shows a HPLC chromatogram illustrating the presence of flavonoidshesperidin, narirutin and neoponcirin in a fraction of collected eluate.

FIG. 7 shows a HPLC chromatogram illustrating the absence of limonoidglucosides in a fraction of an eluate collected after a solutioncontaining limonoid glucosides has passed over an anion exchange resinin accordance with another embodiment of the invention.

FIG. 8 shows a HPLC chromatogram illustrating the presence of limonoidglucosides in an eluate fraction collected after a salt solution hasbeen used to displace the limonoid glucosides from an anion exchangeresin.

FIG. 9 shows a HPLC chromatogram illustrating the presence of limonoidglucosides in a fraction of orange peel extract (OPE) eluted from anacrylic polymer adsorbent (column A) in a process in accordance with oneembodiment of the invention.

FIG. 10 shows a graph illustrating the relative amounts of differentlimonoid glucosides obtained in various fractions of OPE eluted from theacrylic polymer adsorbent (column A) in a process in accordance with oneembodiment of the invention.

FIG. 11 shows a HPLC chromatogram illustrating the presence offlavonoids in an eluate fraction collected after elution of the acrylicpolymer adsorbent (column A) with 40% ethanol in a process in accordancewith one embodiment of the invention.

FIG. 12 shows a graph illustrating the relative amounts of differentflavonoid compounds in various eluate fractions obtained afterdesorption from the acrylic polymer adsorbent (column A) in a process inaccordance with one embodiment of the invention.

FIG. 13 shows a HPLC chromatogram illustrating the absence of flavonoidsand limonoid glucosides in the residual juice fraction obtained afterelution of an OPE fraction from a polystyrene-divinyl benzene polymeradsorbent (column B) in a process in accordance with one embodiment ofthe invention.

FIG. 14 shows a HPLC chromatogram illustrating the presence of limonoidglucoside compounds in an eluate fraction collected after elution of thepolystyrene-divinyl benzene polymer adsorbent (column B) with 30%ethanol in a process in accordance with one embodiment of the invention.

FIG. 15 shows a graph illustrating the relative amounts of differentlimonoid glucoside compounds in various eluate fractions obtained afterdesorption from the polystyrene-divinyl benzene polymer adsorbent(column B) in a process in accordance with one embodiment of theinvention.

FIG. 16 shows a HPLC chromatogram illustrating the presence of limonoidglucoside in an eluate collected after elution of a weak anion exchangeresin (column C) with 0.5M sodium chloride in a process in accordancewith one embodiment of the invention.

FIG. 17 shows a graph illustrating the relative amounts of differentlimonoid glucoside compounds in various eluate fractions obtained afterdesorption from the weak anion exchange resin (column C) in a process inaccordance with one embodiment of the invention.

FIG. 18 shows a HPLC chromatogram illustrating the limonoid glucosidescompounds in eluate fractions collected after elution of apolystyrene-divinyl benzene polymer adsorbent (column D) with 50%ethanol in a process in accordance with one embodiment of the invention.

FIG. 19 shows stacked HPLC chromatograms illustrating the components ina raw orange peel extract (lower line in each chromatogram) and in aconcentrated limonoid glucoside fraction (upper line in eachchromatogram) obtained by a process in accordance with one embodiment ofthe invention.

FIG. 20 is a schematic diagram illustrating a system for carrying out aprocess for selectively separating bioactive compounds in accordancewith one embodiment of the invention.

DETAILED DESCRIPTION

The present invention relates in one aspect to a process for separatingbioactive compounds. The process of the invention enables bioactivecompounds to be selectively separated from one another.

The term “bioactive” as used herein refers to compounds or substancesthat have an effect upon a living organism, tissue or cell. The personskilled in the art would appreciate that bioactive compounds can befound in many food sources and other naturally occurring substances. Inaccordance with one embodiment of the invention, the bioactive compoundsare obtained from vegetable material.

As used herein, the terms “vegetable matter” and “vegetable material”refer to material derived from plants. A range of bioactive compoundsmay be found in a wide variety of different vegetable matter orvegetable material. The vegetable material from which the bioactivecompounds are obtained may be in any form.

In one embodiment the vegetable material is derived from an ediblefruit. Examples of edible fruit include tomatoes, apples, pears, grapes,berries, stone fruit and citrus fruit. Fruits such as grapes may containa number of bioactive compounds including stilbenes such as resveratrol,flavanols such as quercetin and myricetin, catechins and anthocyanins.Other fruits such as apples may also be a source of bioactive compoundssuch as catechins, flavanols and dihydrochalcones. In addition, citrusfruits may contain bioactive limonoids, flavonoids and polymethoxylatedflavones. Vegetable material derived from edible fruit may be obtainedfrom all the parts of the fruit, including the peel, skin, juice,endocarp, seeds and flesh of the fruit.

In another embodiment the vegetable material is derived from plantmaterial that is not an edible fruit. Such plant material includes theflowers, roots, leaves and stems of a plant. Sugarcane is an example ofplant material that is not an edible fruit, which can contain bioactivecompounds such as methoxylated flavones and phenolic acids.

In another embodiment the vegetable material is a vegetable extractwhich is derived from plant material. The vegetable extract is typicallya liquid or solution containing essential components which have beenrecovered from the plant material. An example of a vegetable extract iscitrus peel extract which is derived from citrus peel.

While the following detailed discussion of the invention will be largelyfocussed on bioactive compounds obtained from citrus fruit, it is to beunderstood that the invention is not so limited and is also applicableto bioactive compounds obtained from other plants and plant material. Inparticular, the invention may be used to separate bioactive compounds ofdifferent physical properties.

In one aspect the present invention provides a process for separatingbioactive compounds, the process comprising the step of (a) contacting aplurality of bioactive compounds with a polymer adsorbent underconditions allowing adsorption of at least one bioactive compound on tothe adsorbent while at least one bioactive compound is not adsorbed onto the adsorbent. The plurality of bioactive compounds comprises atleast two, and preferably comprises more than two bioactive compounds.The plurality of bioactive compounds is preferably provided in asolution, which may be prepared using any technique. In one embodiment,the solution is an extract obtained from vegetable material.

In one embodiment, the vegetable material is derived from a citrus fruitsuch as oranges, lemons, limes, grapefruits, mandarins, tangerines andthe like. All parts of the citrus fruit, including the peel and endocarpof the fruit may provide the vegetable material. Preferably, thevegetable material is derived from the citrus peel. The vegetablematerial may also be pre-treated in any suitable manner prior toprocessing in accordance with one aspect of the invention describedherein.

A solution comprising a plurality of bioactive compounds may be obtainedby contacting the vegetable material with a solvent that extracts thebioactive compounds from the vegetable material. Any suitable solventmay be used. A preferred solvent is water. The extraction of thevegetable material by the solvent may proceed by any suitable processknown in the art. For a solution that is an extract derived from citrusfruits, the solution will comprise a mixture of compounds, includinglimonoid and flavanone bioactive compounds, natural sugars and organicacids. If desired, the solution may be subjected to a pre-treatment stepprior to processing in accordance with the invention as describedherein. Centrifugation and filtration are examples of pre-treatmentsthat may be used. Such pre-treatment may be desirable to minimise theamount of suspended solids or other undesirable material in thesolution.

The plurality of bioactive compounds contacts the polymer adsorbent. Thepolymer adsorbent has a selective affinity for at least one of thebioactive compounds present in the mixture. As a result, the bioactivecompound is substantially adsorbed on to the polymer adsorbent andthereby retained by the adsorbent. The polymer adsorbent furthermoredoes not have an affinity for at least one other bioactive compound thatis present in the mixture of bioactive compounds. Consequently, inaccordance with the invention at least one bioactive compound is notadsorbed on to the adsorbent.

Any suitable polymer adsorbent that is capable of selectively adsorbingat least one bioactive compound may be used. Without wishing to belimited by theory, it is believed that favourable interactions, such asionic or hydrogen bonding interactions enhance the ability of a givenbioactive compound to be adsorbed on to the polymer adsorbent. In oneembodiment, the polymer adsorbent is an acrylic. In one embodiment, asuitable polymer adsorbent is an acrylic ester. One example of anacrylic ester is polymethylmethacrylate. The polymethylmethacrylate maybe crosslinked with a suitable crosslinking agent such as ethyleneglycol. It is preferred that the polymer adsorbent be non-ionic. Anexample of a polymer adsorbent suitable for use in the invention isAlimentech P495 Inert Adsorbent Polymer supplied by Bucher Foodtech.

The polymer adsorbent may be provided in any suitable form andarrangement.

In one embodiment, the polymer adsorbent is an acrylic that is providedin the form of beads. The beads may be of any suitable shape or size.The polymer adsorbent may be arranged in any suitable manner. In apreferred embodiment, the polymer adsorbent is arranged in the passage,which may be a column, container, vessel or pipe. Gravity fed columnsand flash chromatography columns are examples of suitable arrangements.Other arrangements, such as moving bed chromatography apparatus, mayalso be used. The ratio of the length of the column to its diameter isat least 4:1, and preferably at least 8:1. The passage may contain anysuitable volume of the adsorbent.

In one embodiment, a solution comprising the plurality of bioactivecompounds is applied to the top of a vertically arranged columncontaining an acrylic polymer adsorbent. While embodiments of theinvention are described herein with reference to the application ofvarious solutions to the top of a vertically arranged column, the personskilled in the art would appreciate that other arrangements, and othermeans of introducing solutions to the arrangements, may also be used.For example, solutions may be fed into the bottom of a verticallyarranged column. Alternatively, if a column is arranged in asubstantially horizontal manner, the solutions may be fed into one endof the horizontal column.

The solution is then allowed to percolate through the adsorbent. Anyquantity of solution may be applied to the absorbent and a personskilled in the relevant art would appreciate that the amount of solutionmay depend on the size of the column as well as the type of adsorbentused. The solution is allowed to pass through the passage at any ratethat enables the solution to sufficiently contact the adsorbent. Aperson skilled in the art would understand that a suitable rate woulddepend on a number of factors, including the size of the apparatus andwhether the process is carried out at a laboratory or industrial scale.

As the solution comprising the plurality of bioactive compounds passesthrough the column containing the polymer adsorbent, at least onebioactive compound is substantially adsorbed on to the polymeradsorbent. The retention of the at least one bioactive compound by theadsorbent removes the compound from the solution.

After the solution has passed through the column, it leaves the columnand is collected. At least one bioactive compound present in thesolution, which has not been adsorbed on to the polymer adsorbent, alsoleaves the column with the solution. The solution that elutes throughand leaves the column is also known as an eluate. Thus the process ofthe invention also comprises the step of (b) collecting a solutioncomprising the at least one bioactive compound which has not adsorbed onto the adsorbent. The at least one bioactive compound which has notadsorbed on to the adsorbent may be collected in a single solutionfraction of eluate or in multiple fractions. Other components that havenot been adsorbed on to the polymer adsorbent may also be present in thecollected fractions. The collected solution (eluate) may be analysed forthe presence of a bioactive compound by any suitable method. A preferredmethod involves the use of high performance liquid chromatography(HPLC). As will be described below, the eluted bioactive compound mayundergo additional treatment to further purify the bioactive compound.

In one embodiment, where the plurality of bioactive compounds contains amixture of flavonoid and limonoid bioactive compounds, the polymeradsorbent is one that is capable of separating the flavonoid andlimonoid compounds. Preferably, the polymer adsorbent is an acrylic,more preferably an acrylic ester and even more preferablypolymethylmethacrylate. An acrylic polymer adsorbent has been found toselectively bind the bioactive flavanones and their derivatives, such asthe flavanone glycosides, more strongly than to limonoid compounds, suchas the limonoid glucosides. This selective adsorption allows theflavanones to be substantially retained by the adsorbent while thelimonoid glucoside compounds are not retained by the adsorbent. In thismanner, the two groups of bioactive compounds are substantiallyseparated. In one form of the method, the two groups of bioactivecompounds are completely separated. Other compounds such as naturalsugars and organic acids also are not retained by the adsorbent. Thuswhere the acrylic adsorbent is arranged in a column, the natural sugarsand organic acids rapidly pass through the column ahead of the limonoidglucosides while the flavonoid compounds do not pass through. Thus inaccordance with one aspect of the invention a flavanone glycoside may besubstantially separated from a limonoid glucoside.

In another embodiment, the plurality of bioactive compounds may alsocontain other bioactive compounds such as the polymethoxylated flavonesand limonoid aglycones in addition to the flavonoid compounds andlimonoid glucosides. In this embodiment, the polymer adsorbent iscapable of adsorbing polymethoxylated flavones and limonoid aglyconesalong with the flavonoid compounds. The method of the inventiontherefore also enables these compounds to be substantially separatedfrom the limonoid glucosides and small molecule polar constituents suchas naturally occurring sugars and organic acids.

In accordance with the one aspect of the invention, the process maycomprise the steps of (c) contacting the at least one bioactive compoundadsorbed on the polymer adsorbent of step (a) with an eluent underconditions allowing desorption of the at least one bioactive compoundfrom the adsorbent, and (d) eluting the at least one bioactive compoundfrom the adsorbent.

When the polymer adsorbent is arranged in a passage such as a column,the eluent is typically introduced in aliquots or as a continuous streamat the top of the column and allowed to percolate through the adsorbent.Where aliquots are introduced, one or more aliquots of eluent may beemployed. The eluent acts to desorb the at least one bioactive compoundfrom the adsorbent and to carry the bioactive compound through thecolumn. The eluent is preferably fed to the passage at a pre-determinedrate, which may vary between 1 and 5 bed volumes per hour andpreferably, is between 1 to 2 bed volumes per hour.

The eluent may comprise any suitable solvent or mixture of solvents.Preferably, the solvent or mixture of solvents is selected from thosepermitted for use in food grade products. A preferred solvent is a watersoluble solvent. In one embodiment, the eluent comprises alcohol andwater.

Where the eluent comprises alcohol, any concentration of alcohol may beused. In one embodiment, the concentration of alcohol in the eluent isin the range of between about 10 to 80% (v/v). A person skilled in theart would understand however that the concentration of alcohol used mayvary depending on the nature of the bioactive compound and the desiredresult. In addition, any suitable alcohol may be employed in the eluent.In one embodiment the alcohol is ethanol.

In one embodiment, the concentration of alcohol in the eluent remainssubstantially constant during desorption of the at least one bioactivecompound from the adsorbent. A preferred alcohol concentration is about40% (v/v). In another embodiment, the concentration of alcohol in theeluent increases during desorption of the at least one bioactivecompound from the adsorbent. Preferably, the alcohol concentrationincreases from about 20% to about 80% (v/v). The concentration ofalcohol may increase at a substantially constant rate or it may increasein a step-wise manner. Where the eluent provides a gradientconcentration of alcohol, aliquots of eluent containing increasingalcohol contents can be sequentially introduced to the top of thecolumn.

Upon leaving the column, the resulting eluate may then be collected infractions. At least one fraction, and preferably multiple fractions,corresponding to the presence of the desorbed bioactive compounds arecollected.

It is emphasised that while fractional collection methods may be usefulin some embodiments of the method, non-fractional collection methods areincluded in the scope of the present invention. For example, a targetbioactive compound may be allowed to adsorb to the polymer adsorbent,while contaminant molecules are allowed to simply pass through thecolumn to waste. After substantially all contaminant molecules areremoved from the polymer adsorbent matrix (for example by washing thematrix with a solution that does not desorb the target bioactivecompound), the target bioactive compound may be desorbed by theapplication of an appropriate solution, and the bulk collection oftarget compound collected in a single volume.

Fractions or bulk collected volumes may be analysed to determine thepresence or amount of bioactive compounds. A preferred analysis methodinvolves the use of High Performance Liquid Chromatography (HPLC). Inone embodiment, where flavanones and their derivatives have adsorbed onto the polymer adsorbent, the collected fraction therefore contains thedesorbed flavonoid compounds.

Where polymethoxylated flavones and limonoid aglycones have also beenadsorbed on to the polymer adsorbent in addition to the bioactiveflavonoid compounds, the eluent used to remove the flavonoid compoundsmay not desorb the polymethoxylated flavones and limonoid aglycones fromthe adsorbent. Accordingly, the polymethoxylated flavones and limonoidaglycones may remain bound to the polymer adsorbent during desorption ofthe flavonoid compounds. Thus, in accordance with another aspect of theinvention a flavonoid compound may be substantially separated from apolymethoxylated flavone and/or a limonoid aglycone.

If desired, after desorption of the flavonoid compounds, the polymeradsorbent may be washed with a suitable solution to remove any adsorbedpolymethoxylated flavones and limonoid aglycones from the polymeradsorbent to re-generate the polymer adsorbent for further use. Anysuitable solution may be used to remove the adsorbed polymethoxylatedflavones and limonoid aglycones from the polymer adsorbent. An exampleof a solution that can be used to desorb the polymethoxylated flavonesand limonoid aglycones is 0.5M sodium hydroxide.

In a further aspect of the invention, the bioactive compound that hasnot adsorbed on to the polymer adsorbent may be further purified. Inaccordance with this aspect of the invention, the process may comprisethe step of (e) contacting the solution comprising the at least onebioactive compound obtained from step (b) with a polymer adsorbent underconditions allowing adsorption of the at least one bioactive compound onto the adsorbent, and (f) collecting the solution eluted from thepolymer adsorbent.

Unlike the polymer adsorbent used previously, the polymer adsorbentemployed in step (e) is selected from any of those that have the abilityto bind to the bioactive compound present in the solution. Preferably,the polymer adsorbent is capable of adsorbing non-polar compounds. Apreferred polymer adsorbent is polystyrene-divinyl benzene. Polarcomponents such as natural sugars and simple organic acids that may alsobe present in the solution are not adsorbed by the polymer adsorbent andare carried with the solution as it leaves the polymer adsorbent and iseluted. An example of a commercially available polystyrene-divinylbenzene polymer adsorbent is Amberlite XAD-16 manufactured by Rohm andHaas.

The polymer adsorbent employed in step (e) may be provided in anysuitable form and arrangement. In one embodiment, the polymer adsorbentis a polystyrene-divinyl benzene polymer in the form of beads. The beadsmay be of any suitable shape or size. The beads may be arranged in anysuitable manner. The beads are preferably arranged in the passage, whichmay be provided by packing the beads in a column, container, vessel orpipe. The passage may contain any suitable volume of the beads. In onepreferred embodiment of the invention, the bed volume of the polymeradsorbent used in step (e) is equivalent that the bed volume of thepolymer adsorbent used in step (a) above. For example, where the bedvolume of the polymer adsorbent used in step (a) is at least about 250litres, the bed volume of the polymer adsorbent employed in step (e) mayalso be at least about 250 litres. However, a person skilled in therelevant art will understand that the volume of polymer adsorbent usedin step (e) may be varied depending on the nature of the bioactivecompounds and the desired result.

In one embodiment, the solution obtained from step (b), which comprisesthe at least one bioactive compound, is applied to the top of a columncontaining the polymer adsorbent and allowed to percolate through theadsorbent in order to contact the polymer adsorbent in step (e). In oneembodiment, when the solution comprises bioactive limonoid compounds andderivatives such as limonoid glucosides, the limonoids bind to thepolymer adsorbent during passage of the solution through the adsorbentand are thereby removed from the solution. The solution traverses thepassage and upon leaving the passage and polymer adsorbent, is collectedas an eluate. In one embodiment, where the collected solution (oreluate) contains sugars and simple organic acids, the eluted solutionmay be regarded as a purified “juice” component. It has been found thatthe juice obtained in accordance with the process of the invention isneutral and not bitter. It is contemplated that the purified “juice”component may be isolated and, if desired, further treated for use as asupplement in food products such as beverages.

In accordance with one embodiment of the invention, once the solutionhas eluted from the adsorbent, the at least one bioactive compound maybe desorbed from the polymer adsorbent of step (e). Accordingly, thepresent invention may further comprise the step of (g) contacting the atleast one bioactive compound adsorbed on the polymer adsorbent employedin step (e) with an eluent under conditions allowing desorption of theat least one bioactive compound from the adsorbent, and (h) eluting theat least one bioactive compound from the adsorbent. One or more aliquotsof eluent, or a continuous stream of eluent, may be used to desorb theat least one bioactive compound from the polymer adsorbent.

Similar to the eluent used to desorb bioactive compounds from thepolymer adsorbent in step (c) as described above, the eluent employed instep (g) may comprise any suitable solvent or mixture of solvents.Preferably, the solvent or mixture of solvents is selected from thosepermitted for use with food products. In one embodiment, the eluentcomprises alcohol and water. Where the eluent comprises alcohol, anysuitable concentration of alcohol may be used. In one embodiment, theconcentration of alcohol in the eluent is in the range of from about 10to 80% (v/v). A person skilled in the art would understand however thatthe concentration of alcohol used may vary depending on the nature ofthe bioactive compound and the desired result. In addition, any type ofalcohol may be used. A preferred alcohol is ethanol.

In one embodiment, the concentration of alcohol in the eluent remainssubstantially constant during desorption of the at least one bioactivecompound from the adsorbent. Preferably, the eluent comprises about 40%(v/v) alcohol. In another embodiment, the concentration of alcohol inthe eluent increases during desorption of the at least one bioactivecompound from the adsorbent. Preferably, the alcohol concentrationincreases from about 10% to about 80% (v/v). The concentration ofalcohol may increase at a substantially constant rate or it may increasein a step-wise manner. Where the eluent provides a gradientconcentration of alcohol, aliquots of eluent containing increasingalcohol contents can be sequentially introduced to the top of a columncontaining the polymer adsorbent.

The bioactive compound eluted from the polymer adsorbent in accordancewith step (h) is typically collected in an eluate fraction. The eluatefraction is a solution that comprises the bioactive compound togetherwith the solvent used to elute the bioactive compound from the polymeradsorbent. At least one fraction, and preferably a plurality of elutedfractions, corresponding to the presence of the bioactive compound arecollected. In one preferred embodiment of the invention, the collectedeluate fraction contains limonoid compounds such as limonoid glucosides.Where more than one eluate fraction is collected, the eluate fractionsmay be combined to form a single fraction. In addition, as discussedabove, non-fractional collection methods which enable the targetbioactive molecule to be collected in a single bulk volume may also beused if desired.

In the case of the limonoids, the use of an eluent in which theconcentration of alcohol increases during desorption of the limonoidsmay be advantageous where it is desired to separate selected limonoidcompounds from each other. Different limonoid compounds possessdifferent polarities due to differences in chemical structure.Accordingly, the use of an alcohol gradient may assist to separate thelimonoid compounds based on differences in polarity. In terms of orderof elution as the concentration of alcohol in the eluent increases,limonin glucoside, which is the most polar compound, is eluted firstfrom the polymer adsorbent, followed by nomilin glucoside, nomilinicacid glucoside and obacunone glucoside. A person skilled in the relevantart would be able to adjust the alcohol concentration in the eluent anddetermine whether the desired separation is achieved by monitoring theelution of the limonoid compounds using analytical methods such as HPLC.

In some embodiments of the invention, the bioactive compounds that areeluted from the polymer adsorbent may be further treated to purify andconcentrate the bioactive compounds.

In another aspect, the present invention relates to a process forpurifying a bioactive compound, the process comprising the step ofcontacting the bioactive compound with an ion exchange resin underconditions allowing ionic interactions between the bioactive compoundand the resin such that the bioactive compound is adsorbed on to theresin.

Where at least one bioactive compound has been eluted from a polymeradsorbent in accordance with the process of one aspect of the invention,the process may further comprise the step of contacting the elutedbioactive compound with an ion exchange resin under conditions allowingionic interactions between the at least one bioactive compound and theresin such that the at least one bioactive compound is adsorbed on tothe resin.

In one embodiment, the bioactive compound to be purified is a bioactiveobtained after elution from a polymer adsorbent, in accordance with step(h) described above. In this embodiment, the bioactive compound istypically provided in a solution with the elution solvent. It is anadvantage of the present invention that the solution (or eluate)comprising the bioactive compound can be directly applied to the ionexchange resin without the need to remove excess alcohol from thesolution by evaporative or other processes prior to exposure of thebioactive compound to the ion exchange resin.

In one embodiment the ion exchange resin is an anionic exchange resinand preferably, is a weak anion exchange resin. An example of a weakanion exchange resin that may be used in the present invention is theDiaion WA-30 resin supplied by Supelco.

The ion exchange resin may be provided in any suitable form andarrangement. A range of suitable forms and arrangements would beapparent to a person skilled in the relevant art. The resin may bearranged in the passage, which may be provided by packing the resin in acolumn such as a gravity fed column or a flash chromatography column.The passage may contain any suitable volume of the resin. In oneembodiment, the bed volume of the ion exchange resin is about 20% of thebed volume of polymer adsorbent used in step (a) above. For example, if100 litres of polymer adsorbent is used in step (a), then 20 litres ofion-exchange resin may only be required. A person skilled in the artwill however, understand that the required volume of ion exchange resinmay vary depending on the nature of the bioactive compounds and thedesired result.

In one embodiment, a solution containing the bioactive compound isintroduced to the top of the resin contained in a column and allowed topercolate through the resin. In this manner, contact between thebioactive compound and the ion exchange resin is achieved.

It is believed that ionic interactions between the resin and bioactivecompounds such as the limonoid glucosides lead to the selectiveadsorption of the bioactive compounds on to the resin. Other componentsthat may be present that are unable to participate in the ionicinteractions will not bind to the resin. Accordingly, the bioactivecompounds may be concentrated and further separated from any undesirablecomponents which may contaminate the bioactive compounds.

Bioactive compounds that have adsorbed on to the ion exchange resin maybe recovered by contacting the ion exchange resin with a solutioncomprising a suitable solute under conditions allowing displacement ofthe bioactive compound from the resin. The solute may be selected fromany of those that are able to compete with the bioactive compound forbinding sites in the ion exchange resin. A preferred solute is a salt,such as sodium chloride. The solute may be present in any suitableconcentration. In one embodiment, the solution is a 0.5M sodium chloridesolution.

In one embodiment, where the bioactive compound is a limonoid glucoside,a solute solution comprising a salt as the solute may be passed througha passage containing the ion exchange resin. One or more aliquots of thesolute solution, or a continuous stream of the solute solution, may beintroduced to the passage containing the ion exchange resin. The saltcompetitively binds to the resin and displaces the limonoid glucosidefrom the resin. The desorbed limonoid glucosides are subsequentlycollected as volumes of the solute solution leaves the passage. Thedesorbed limonoid glucosides may be collected in a single fraction ormultiple fractions, or in a bulk volume of the eluted solute solution.

After collection of the bioactive compound, any solute that is presentin the collected fractions is preferably removed. The solute may beremoved by any suitable process. In a preferred embodiment, the soluteis removed by contacting the fractions with a polymer adsorbent. Thefractions are contacted with the adsorbent under conditions allowingadsorption of the bioactive compound on to the adsorbent.

Any suitable polymer adsorbent may be used. Preferably, the polymeradsorbent is capable of adsorbing non-polar compounds. A preferredpolymer adsorbent is polystyrene-divinyl benzene. An example of asuitable polystyrene-divinyl benzene polymer adsorbent is AmberliteXAD-16 manufactured by Rohm and Haas. Similar to the polymer adsorbentsdescribed above, the adsorbent may be provided as beads in a passagesuch as a column. Any suitable volume of the polymer adsorbent may beused. In one embodiment, the bed volume of the polystyrene-divinylbenzene polymer resin may be about 20% of the bed volume of polymeradsorbent used in step (a) above. A person skilled in the art howeverwill understand that the required volume of polymer resin may varydepending on the nature of the bioactive compounds and the desiredresult.

In one embodiment, a collected volume of solute solution comprising thebioactive compound is applied to the top of the column and allowed topercolate through the adsorbent. Where the bioactive compound compriseslimonoid compounds, the limonoids bind to the polymer adsorbent and aresubstantially retained by the polymer adsorbent. Meanwhile, the solutedoes not bind to the adsorbent and is eluted from the adsorbent. In thismanner, the limonoid bioactive compounds are separated from theundesirable solute.

The bioactive compound may then be desorbed from the polymer adsorbentby contacting the polymer adsorbent with an eluent and eluting thebioactive compound from the adsorbent in accordance with previouslydescribed procedures. A preferred eluent comprises a mixture of alcoholand water. Any suitable concentration of alcohol may be used. In oneembodiment, the eluent comprises alcohol in an amount in the range fromabout 10 to about 80% (v/v). In addition, any suitable alcohol may beused. A preferred alcohol is ethanol.

The concentration of alcohol in the eluent may remain substantiallyconstant. In a preferred embodiment, the eluent comprises at least about40% (v/v) alcohol and more preferably, comprises about 70% (v/v)alcohol. Alternatively, the concentration of alcohol may increase duringdesorption of at least one bioactive compound from the adsorbent. Wherethe concentration of alcohol increases during desorption of thebioactive compound, the alcohol content may increase at a substantiallyconstant rate or in a step-wise manner. In one embodiment, theconcentration of alcohol in the eluent may increase from about 10 to 80%(v/v) during desorption of at least one bioactive compound from theadsorbent.

The eluted bioactive compound is subsequently collected in at least one,and preferably in multiple fractions corresponding to the presence ofthe bioactive compound. The presence of the bioactive compound may beanalysed by any suitable method. An example of a suitable method isHPLC. The obtained bioactive compound has been found to be of higherpurity than bioactives prepared using prior art processes.

In another aspect, the present invention relates to a purified bioactivecompound prepared by a process as described herein. In one embodiment,the bioactive compound is a limonoid compound, more preferably alimonoid glucoside. In another embodiment, the purified bioactivecompound is a flavonoid compound, preferably a flavonone glycoside.

In yet another aspect, the present invention relates to a process forthe selective extraction of bioactive compounds from a vegetablematerial, the process comprising the step of contacting the vegetablematerial with a solvent under conditions allowing extraction of at leastone water soluble bioactive compound from the vegetable material tothereby provide an extract comprising the water soluble bioactivecompound and a vegetable residue comprising the at least one waterinsoluble bioactive compound. In a preferred embodiment, the solventused to contact the vegetable material is water. Accordingly in thisembodiment, an aqueous extract containing the at least one water solublebioactive compound is formed.

The term “vegetable material” is used herein to refer to materialderived from plants. The vegetable material may be in any form asdescribed herein. The vegetable material may also be pre-treated in anysuitable manner prior to processing in accordance with the invention asdescribed herein. In one embodiment the vegetable material may bederived from an edible fruit, such as those described herein. Thevegetable material may be derived from a citrus fruit such as oranges,lemons, limes, grapefruits, mandarins, tangerines and the like. Allparts of the citrus fruit, including the peel and endocarp of the fruitmay provide the vegetable material. In one embodiment, the vegetablematerial may be derived from the citrus peel. The vegetable material mayalso be a vegetable extract derived from plant material.

The solvent may contact the vegetable material using any suitabletechnique. In one embodiment, a counter-current extractor is used tocontact the solvent with the vegetable material and thereby extract theat least one water soluble bioactive compound from the vegetablematerial. In another embodiment, the vegetable material is treated inmanner that results in the vegetable material being placed in a finelydivided form. The digested vegetable material is then run through aroller to squeeze out juices containing water soluble bioactivecompounds. In one embodiment, the vegetable material comprises limonoidssuch as limonoid glucosides and flavonoid compounds such as flavanoneglycosides as water soluble bioactive compounds.

The extraction of the water soluble bioactive materials from thevegetable material may be performed under any suitable conditions. Inone embodiment, the vegetable material is treated at a temperature of atleast 70° C., typically for a short period. In one embodiment of themethod, the vegetable material is treated at a temperature of at least70° C. for about 2 minutes. Without wishing to be limited by theory, itis thought that the high temperature treatment step assists to destroyoxidative enzymes and microbes, which may be detrimental to the desiredend product. The high temperature may also assist in the disruption ofthe cellular structure of the vegetable material, enabling solublecompounds in the vegetable material to diffuse into the counter-flowingsolvent. Following this, the temperature of the vegetable material maythen be lowered to optimize the extraction of the desirable compounds.

After extraction of the water soluble bioactive compounds from thevegetable material, a vegetable residue remains. The vegetable residuemay comprise at least one water insoluble bioactive compound, which isnot removed by the initial solvent extraction procedure. The vegetableresidue may be in the form of a solid or a liquid, depending on theinitial form of the vegetable material.

In another aspect of the invention, the resulting vegetable residue maybe contacted with an extraction solution comprising alcohol in order toextract at least one water insoluble bioactive compounds from thevegetable residue. The solution may contact the vegetable residue forany time and under any conditions sufficient to extract at least onewater insoluble bioactive compound from the residue and thereby providean alcoholic extract comprising the at least one water insolublebioactive compound. In one embodiment, the vegetable material and hencethe vegetable residue comprises polymethoxylated flavones as the waterinsoluble bioactive compounds.

Any suitable technique may be used to contact the extraction solutionwith the vegetable residue and thereby extract at least one waterinsoluble bioactive compound from the residue. In one preferredembodiment, a counter-current extractor is used.

The extraction solution that contacts the vegetable residue may compriseany suitable amount of alcohol. In one embodiment, the extractionsolution comprises a mixture of water and alcohol. Preferably, theextraction solution comprises at least about 10% alcohol and morepreferably at least about 20% alcohol. Any suitable alcohol may be used.A preferred alcohol is ethanol.

If required, the alcoholic extract containing the at least one waterinsoluble bioactive compound may be subsequently contacted with apolymer adsorbent under conditions allowing adsorption of the waterinsoluble bioactive compound on to the adsorbent. This assists tofurther purify the water insoluble bioactive compounds by separating thewater insoluble bioactive compounds from any impurities that are notable to be retained by the adsorbent. The polymer absorbent ispreferably one that adsorbs to non-polar compounds. A preferred polymeradsorbent is polystyrene-divinyl benzene. An example of a suitablepolystyrene-divinyl benzene polymer adsorbent is Amberlite XAD-16manufactured by Rohm and Haas.

The polymer adsorbent may be provided in any suitable form andarrangement. In one embodiment, the polymer adsorbent is apolystyrene-divinyl benzene polymer in the form of beads. The beads maybe of any suitable shape or size. The beads may be arranged in thepassage, which may be provided by packing the beads in a column,container, vessel or pipe. Gravity fed columns and flash chromatographycolumns are examples of suitable columns. The passage may contain anysuitable volume of the beads. A person skilled in the art wouldunderstand that the volume of polymer adsorbent used may depend upon anumber of factors, such as for example, the amount of material to beapplied to the adsorbent. Other arrangements, such a moving bedchromatography apparatus, may also be used.

In one embodiment, the alcoholic extract comprising the at least onewater insoluble bioactive compound is introduced to the top of a columncomprising the adsorbent and allowed to percolate through the adsorbent.In this manner, the at least one water insoluble bioactive compound areable to contact the polymer adsorbent. The water insoluble bioactivecompound, being generally non-polar in nature, is retained by theadsorbent while polar components that may be present in the extract arenot retained and pass through the passage and are collected. In oneembodiment the at least one water insoluble bioactive compound comprisespolymethoxylated flavones. Thus, the polymethoxylated flavones areadsorbed on to the polymer adsorbent.

Water insoluble bioactive compounds may be subsequently removed from theadsorbent by contacting the adsorbent with an eluent under conditionsallowing desorption of the water insoluble bioactive compounds from theadsorbent, and eluting the water insoluble bioactive compounds from theadsorbent.

Where the polymer adsorbent is arranged in a column, the eluent may beintroduced in aliquots, or in a continuous stream, at the top of thecolumn and allowed to percolate through the adsorbent. Where the eluentprovides a gradient concentration of alcohol, aliquots of eluentcontaining increasing alcohol contents can be sequentially introduced tothe top of the column. The eluent acts to desorb the bioactive compoundsfrom the adsorbent and carry the bioactive compounds through the column.The eluent is preferably fed to the passage at a pre-determined rate,which may vary between 1 and 5 bed volumes per hour. A person skilled inthe art would understand however, that a suitable rate would depend onnumber of factors, including the size of the apparatus and whether theprocess is carried out at a laboratory or industrial scale.

The eluent may comprise any suitable solvent or mixture of solvents.Preferably, the solvent or mixture of solvents is selected from thosepermitted for use in food products. In one embodiment, the eluentcomprises alcohol and water. Where the eluent comprises alcohol, anyconcentration of alcohol may be used. Preferred alcohol concentrationsare in the range of from about 10 to 80% (v/v). However, it would beappreciated by the skilled addressee that the concentration of alcoholused may vary depending on the nature of the bioactive compound and thedesired result. In addition, any suitable alcohol may be employed in theeluent. A preferred alcohol is ethanol.

The concentration of alcohol in the eluent may remain substantiallyconstant. In a preferred embodiment, the eluent comprises at least about40% (v/v) alcohol, preferably at least about 50% alcohol and morepreferably, about 70% (v/v) alcohol. Alternatively, the concentration ofalcohol may increase during desorption of the at least one bioactivecompound from the adsorbent. The alcohol content may increase at asubstantially constant rate or in a step-wise manner. In one embodiment,the concentration of alcohol in the eluent may increase from about 10 to80% (v/v) during desorption of at least one bioactive compound from theadsorbent.

Upon leaving the column, the eluent is then collected. At least onefraction, and preferably multiple fractions, of eluent corresponding tothe presence of the desorbed water insoluble bioactive compounds arecollected. Furthermore, non-fractional collection methods may also beemployed. The collected solution may be analysed to determine thepresence of bioactive compounds. A preferred analysis method involvesthe use of HPLC.

In one embodiment the water insoluble bioactive compounds derived fromthe vegetable material and the vegetable residue comprisespolymethoxylated flavones. In one embodiment therefore, the fractioncollected from the polymer adsorbent contains the desorbedpolymethoxylated flavone compounds. The polymethoxylated flavones havebeen separated from other bioactive compounds present in the vegetablematerial.

In a further aspect, the present invention relates to purifiedpolymethoxylated flavones prepared by a process as described herein.

Referring now to FIG. 20, a schematic diagram of a system for carryingout a process in accordance with one embodiment of the invention isshown. In this embodiment, a vegetable extract obtained from citrus peelis fed into a counter current extractor (1) and contacted with water toextract water-soluble components from the citrus peel extract. The watersoluble components, which include bioactive flavonoid and limonoidcompounds, are isolated in an aqueous extract. The aqueous extract isthen fed to a filter (2) to remove any solid material from the extract.The filtered aqueous extract is loaded onto a column packed with anacrylic polymer adsorbent (3). The flavonoid compounds are adsorbed ontothe acrylic adsorbent (3) while the limonoid compounds, which do notsubstantially adhere to the acrylic adsorbent (3), pass through theadsorbent (3) and are collected.

The adsorbed flavonoid compounds are desorbed from the acrylic adsorbent(3) by passing an eluent containing 60% ethanol in water through thecolumn (3). Eluate fractions corresponding to the desorbed flavonoidcompounds are then collected. The collected fractions are passed on toalcohol recovery evaporators (4) which remove most of the ethanol toallow collection of the purified flavonoid compounds. The removedethanol can be stored in an ethanol tank and subsequently distilled forre-use if desired.

A solution containing the limonoid compounds which have eluted from theacrylic polymer adsorbent (3) is then loaded onto a column packed with apolystyrene-divinyl benzene polymer adsorbent (5). The limonoidcompounds (and possibly some other non-polar compounds such asflavonoids) are adsorbed onto the polystyrene-divinyl benzene adsorbent(5) while polar components such as the natural sugars and simple organicacids are not adsorbed and are eluted from the polymer adsorbent (5).The eluted solution, which contains the natural sugars and simpleorganic acids, forms a purified “juice” component and is subsequentlycollected. An eluent comprising 30% ethanol in water is then used toremove the adsorbed limonoid compounds from the polystyrene-divinylbenzene polymer adsorbent (5) and fractions corresponding to thepresence of the desorbed limonoid compounds are collected.

After desorption from the polystyrene-divinyl benzene polymer adsorbent(5), the fractions containing the limonoid compounds are then loadedonto a column containing an anion exchange resin (6). The anion exchangeresin (6) adsorbs the limonoid compounds while any components that donot bind to the anion exchange resin pass through the column containingresin (6) and are collected. A salt solution is then loaded onto theanion exchange resin (6) to desorb the limonoid compounds from the resin(6). Fractions containing the desorbed limonoid compounds as well as thesalt are collected. The collected fractions correspond to the presenceof the limonoid compounds.

The fractions containing the limonoid compounds desorbed from the anionexchange resin (6) are then subsequently loaded onto a column containinga polystyrene-divinyl benzene polymer adsorbent (7). The polymeradsorbent (7) is used to remove the salt present in the solution withthe limonoid compounds. The limonoid compounds adsorb on to thepolystyrene-divinyl benzene polymer adsorbent (7) while the salt, whichis not adsorbed, passes through the column containing the polymeradsorbent (7). The limonoid compounds are then desorbed from the polymeradsorbent (7) by passing an alcoholic solution through the adsorbent(7). Eluate fractions corresponding to the presence of the desorbedlimonoid compounds are then subsequently collected.

The collected fractions comprising the desorbed limonoid compounds maybe passed on to alcohol recovery evaporators (4) to remove the ethanolfrom the fractions and allow collection of the purified limonoidcompounds. The removed ethanol can be stored in an ethanol tank andsubsequently distilled for re-use if desired.

After extraction of the water soluble components, the citrus peelextract can be further extracted by feeding the citrus peel extract to asecond counter-current extractor (8) and contacting with a 10% aqueousethanol solution in order to extract water-insoluble compounds from thepeel extract. This process provides an alcoholic extract comprisingwater-insoluble compounds, including polymethoxylated flavone bioactivecompounds.

The alcoholic extract is loaded onto a column containing apolystyrene-divinyl benzene polymer adsorbent (9). Non-polar waterinsoluble compounds such as the polymethoxylated flavones adsorb ontothe polystyrene-divinyl benzene adsorbent (9) and are retained by theadsorbent while any components that are not able to adsorb onto thepolymer adsorbent pass through the adsorbent (9).

The adsorbed polymethoxylated flavones are subsequently removed from thepolystyrene-divinyl benzene adsorbent (9) by passing a 96% ethanol andwater solution through the column. The desorbed polymethoxylated flavonecompounds are then collected in eluate fractions corresponding to thepresence of the bioactive compounds. The collected eluate fractions maybe passed on to alcohol recovery evaporators (10) which remove theethanol to allow collection of the purified polymethoxylated flavones.If desired, the removed ethanol can be stored in an ethanol tank andsubsequently distilled for re-use.

The above system may be operated as a continuous process or in abatch-wise manner. In a continuous process, columns containing each ofthe required polymer adsorbents and the anion exchange resin may bearranged in a sequence, such that once the desired bioactive compoundsare eluted from one column, the resultant eluate is directly fed ontothe subsequent column.

The bioactive compounds obtained in accordance with the presentinvention may also be treated in any suitable manner that facilitatesthe subsequent use or storage of the compounds. In one preferredembodiment, the bioactive compounds may be subjected to evaporativeprocesses, such as for example freeze-drying, to remove excess solventfrom the compounds and thereby place the bioactive compounds in anappropriate form for storage or further use.

Yet a further aspect of the present invention provides a bioactivecompound produced by a process as described herein. A further aspectprovides a composition comprising a limonoid glycoside. The presentinvention may significantly enhance the purity of recovered of bioactivecompounds from vegetable materials and vegetable extracts. The inventionmay also lead to increased recoveries of purified bioactive compounds.For example, limonoid glucosides are able to be recovered from citrusfruits at a concentration of about 50% to 70% on a dry weight basis.This compares favourably to processes of the prior art in which limonoidglucosides can typically be recovered in amounts which may be as low asabout 10% to 15%. In addition, the process of the inventionadvantageously provides improved purity of the recovered bioactivecompounds. Accordingly, polymethoxylated flavones can be recovered withless mixing with the bitter principle limonin.

Because bioactive compounds prepared in accordance with the process ofthe invention are of improved purity they are more easily formulatedinto functional foods allowing much smaller overall doses to provide aneffective dose of the target bioactive and hence a reduced likelihood ofundesirable flavours imparted in the food formulation.

EXAMPLES

The following examples illustrate the present invention in furtherdetail however the examples should by no means be construed as limitingthe scope of the invention as described herein.

Materials and Methods Orange Peel Extract

Orange peel extract (OPE) was supplied as a 20.4 Brix solution by LangTechnologies. Brix was determined by measuring specific gravity and useof conversion tables. Briefly, the mass of 25 ml of OPE was measured onan electronic balance. The specific gravity was determined by dividingthis mass by that of the same volume of de-ionized water and was foundto be 1.085. From conversion tables, this is equivalent to 20.4 Brix.The pH of the OPE was about 4.0 (Merck Universal Indicator paper). FIG.1 shows a HPLC chromatogram of a representative sample of orange peelextract.

Alcohol

Alcohol used in the eluting solvents was supplied as undenatured 95%ethanol.

High Performance Liquid Chromatography (HPLC)

HPLC was used to monitor the progress of the elution of bioactivecompounds from the adsorbent polymer resins. HPLC was performed underthe following conditions:

-   -   Apparatus: Shimadzu VP7 HPLC system consisting of low-pressure        mixing system, SPD-M10A VP diode array detector and VP software        to control gradient and detector.    -   Mobile Phase D: 0.1% (v/v) Aqueous Phosphoric Acid.    -   Mobile Phase A: Acetonitrile    -   Run Time 55 minutes    -   Column: Alltima C1 8 5u Part Number 88056 with guard column    -   Monitoring Wavelength 210 and 280 nm (data collected between 200        nm and 350 nm in 2 nm steps).    -   Oven Temperature: 30° C.    -   Flow Rate: 1.0 ml/min (back pressure 2915 kgf/cm²).    -   Injection Volume 20 μl    -   The HPLC gradient used to determine the presence of limonoid        compounds is shown in Table 1.

TABLE 1 HPLC Gradient used to determine limonoid glycosides. Time(minutes) Solvent D Conc. 0.01 90.0% 35.00 70.0% 45.00 60.0% 46.00 90.0%55.00 Stop

Column back pressure was monitored from run to run to ensure thatperformance was the same.

Example 1 Separation of Bioactive Compound with Acrylic PolymerAdsorbent

A glass column internal diameter 40 mm and height 540 mm fitted with aTeflon tap was partially filled to a height of 350 mm with an ethyleneglycol crosslinked polymethylmethacrylate absorbent polymer resin innon-ionic form (CAS 25777-18-5) (supplied as Alimentech P495 InertAbsorbent Polymer by Bucher Foodtech). The resin bed volume was 400 mlwith an interstitial dead volume of 180 ml. The acrylic polymeradsorbent was conditioned by washing with ten bed-volumes of water priorto use.

Three 250 ml aliquots of OPE (750 ml OPE in total) were applied to thetop of the column containing the acrylic polymer adsorbent, with eachaliquot allowed to percolate down the column at a rate of 4 ml/minute.At this rate each aliquot took about an hour to elute through thecolumn. After application of the OPE, the column was washed with two 250ml aliquots of water.

An eluting solvent containing a mixture of alcohol and water was thenapplied to the top of the column in 250 ml aliquots. The concentrationof alcohol in the eluting solvent increased with each aliquot applied.The eluting solvent passed through the column and was subsequentlycollected in fractions and analysed by HPLC.

It is noted that the alcoholic strength of the eluted fractions may besomewhat less than that applied to the top of the column as the latteradmixes with the liquid remaining within the interstitial “dead volume”of the polymer adsorbent. It is estimated that the concentration ofalcohol is lower by approximately one third of the difference betweenthe strength of the current eluate fraction and the prior one it isreplacing. Thus, for example, if a 20% solution was added after a 10%solution, the concentration of alcohol in the eluate would be around17%.

A total of twelve fractions were collected for analysis by HPLC. Theirdescriptions appear in Table 2.

TABLE 2 Fraction Identity 1 Eluate after First 250 mL OPE applied 2Eluate after Second 250 mL OPE applied 3 Eluate after Third 250 mL OPEapplied 4 First Water Wash 5 Second Water Wash 6 10% Ethanol in water 720% Ethanol in water 8 30% Ethanol in water 9 40% Ethanol in water 1050% Ethanol in water 11 60% Ethanol in water 12 80% Ethanol in water

Results

Chromatograms corresponding to the results of HPLC analysis of the abovefractions are shown in FIGS. 2 to 6. The components of the OPE wereidentified by retention time. The Figures show that a variety ofcomponents are present in the fractions leaving the column.

The chromatogram obtained from fraction 1 is shown in FIG. 2. As seen inFIG. 2, the peaks are small, with most material eluting within fourminutes. The first peak at 2 minutes corresponds to sugars, and thesecond at 4 minutes is phlorin, a major constituent of orange peelextract. Similar results were also obtained for fractions 2 to 6.

In fraction 7, the peaks are starting to increase in size as shown inFIG. 3. The peak at 25.48 is limonin glucoside (LG).

In fractions 8 and 9, the peak corresponding to limonin glucoside wasobserved to increase. In addition, as seen in the chromatogram obtainedfor fraction 9 (FIG. 4), four peaks were observed with a typicallimonoid conformance, namely limonin glucoside (25.39 minutes), arelated limonoid (29.9 minutes), nomilin glucoside (NG) (34.16 minutes)and obacunone glucoside (OG) at 36.98 minutes.

In fraction 10, the limonoid at 34 minutes was observed to be stillsignificant but those eluting earlier have decreased significantly insize.

As the alcohol concentration increases to around 60% in fraction 11,flavonoid compounds begin to elute as shown in FIG. 5. The two majorpeaks here are the flavonoids hesperidin (28.13 minutes) and narirutin(30.36 minutes).

In fraction 12, the alcohol concentration is around 80%. As seen in FIG.6, the predominant compounds in fraction 12 are hesperidin and narirutinwith another flavonoid at 40.04 minutes considered to be neoponcirin(didymin).

Determination of Limonin Glucoside Concentration

As commercial limonin glucoside standards are not available, theconcentration of limonoid glucosides was determined against limonin(Sigma Aldrich) by comparison of peak areas and application of aconversion value (1.4) to allow for the molecular weight of glucose.

A linearity check of the HPLV system was carried out using limoninstandards. The results of the linearity check and calibration are shownin Table 3.

TABLE 3 Results of linearity check and calibration Concentration PeakArea 210 nM 150 9848525 300 19645424 450 2935005 600 3918728 750 4886204

A calibration chart of concentration vs peak area (at 210 nm) wasconstructed for limonin and used to determine the limonoid content ofthe collected fractions.

The raw data of limonoid content in fractions 7-10 is shown in Table 4together with the limonoid content of a Comparative Example:

TABLE 4 Raw Data (Area) Peak Areas Fraction LG NG OG 7 2459092 245909 84427660 442766 1869183 9 7236151 1269381 2532814 10 2531755 ComparativeExample 7022709 ND

By comparison with the calibration chart, an estimate of the amount oflimonoid glucoside in the orange peel extract may be obtained. Theresults are shown in Table 5.

TABLE 5 Concentrations of LG's in OPE (ppm) Fraction LG NG OG 7 375 36 8679 66 286 9 1110 193 387 10 387 Comparative Example 1077

NG and OG were not determined in the original because of interferencesdue to other compounds.

As seen in this Example, the limonoids appear to be less stronglyretained on the acrylic polymer resin and eluted from the acrylic resinat a lower alcohol concentration than the flavonoid compounds. Thisallowed the limonoid compounds to be separated from the flavonoidcompounds without significant cross-contamination of the collectedfractions.

Example 2 Separation of Bioactive Compound with Anion Exchange ResinColumn

A column of 2 cm diameter and 20 cm in length was filled to a height of200 mm with a weak anion exchange resin (Diaion WA-30 resin averageparticle size 0.47 mm, total exchange capacity 1.5 meq/mL supplied bySupelco). The resin bed volume was 50 mL. The anion exchange resin(WA-30) was conditioned prior to use by washing with 2 bed-volumes (100mL) of 0.5M sodium hydroxide, followed by five bed volumes (250 mL) ofwater, followed by 2 bed volumes of 0.5M hydrochloric acid, followed byfive bed volumes of water. The pH of the final washing water was 4.2.

Fractions 8 and 9 from Example 1 were treated to further purify thelimonoid glucosides in these fractions. These fractions, which 30 and40% alcohol respectively, conveniently contain a high proportion ofLG's.

Fraction 8 was poured on to the top of conditioned weak anion exchange(WA-30) resin. The solution was allowed to pass through the column andthe eluate was collected and analysed by HPLC.

In a similar manner, fraction 9 was poured on to the top of the WA-30resin and the eluate was similarly collected and analysed by HPLC.

The anion exchange resin was then subsequently washed with 250 ml of 30%alcohol. Three 150 mL aliquots of 0.5M NaCl were then passed through theanion exchange resin and fractions of the eluted salt solution werecollected and analysed by HPLC.

Results

Table 6 lists each of the analyses undertaken by HPLC.

TABLE 6 HPLC Gradient used to determine limonoids. Time Solvent D Conc.0.01 90.0 35.00 70.0 45.00 60.0 46 90.0 55.00 Stop

A chromatogram of the eluate obtained after application of fraction 9 onto the WA-30 column is shown in FIG. 7. The peaks corresponding to thelimonoids in fraction 9, which were previously observed in FIG. 4, areabsent because the WA-30 resin has retained these compounds. Othercomponents have not been retained by the resin and have eluted from thecolumn. A similar result was observed after fraction 8 was applied tothe WA-30 resin. This shows that the limonoid compounds are bound to theWA-30 resin.

The passage of aliquots of 0.5M NaCl over the WA-30 resin was used todesorb the limonoid compounds from the resin. A chromatogram of aneluate obtained after passing 300 ml of 0.5M NaCl over the WA-30 resinis shown in FIG. 8. As seen in FIG. 8, the eluate fraction is rich inLG's. LG's do not have a UV extinction coefficient as high as theflavonoids, so the purity of LG's in this fraction appears to be greaterthan 75%.

This result shows that limonoid glucosides can be purified byselectively adsorbing the LG's on to an ion exchange resin. A saltsolution is then able to effectively remove the bound limonoidglucosides from the resin. The resultant purified limonoid glucosidesappear to have a purity in excess of 50%. In addition, the purifiedlimonoid glucosides are not contaminated by sugars, or flavonoids suchas narirutin, hesperidin and neoponcirin.

Example 3 Process for Recovering Bioactive Compounds from Orange PeelExtract (OPE)

A process for separating bioactive compounds from orange peel extract(OPE) is described. The process used four separate columns as follows:

Column A: The column was glass with internal diameter 40 mm and height540 mm fitted with a Teflon tap. It was partially filled to a height of200 mm with acrylic resin (Alimentech P495 Inert Absorbent Polymersupplied by Bucher Foodtech). The resin bed volume was 250 mL and theinterstitial dead volume was 110 mL.

Column B: The column was glass with internal diameter 23 mm and height180 mm fitted with a Teflon tap. It was partially filled to a height of180 mm with polystyrene-divinylbenzene resin (Amberlite XAD-16 SurfaceArea 800 m2/g, Average Pore Diameter 100 Angstroms supplied bySigma-Aldrich). The resin bed volume was 50 mL.

Column C: The column was glass with internal diameter 23 mm and height180 mm fitted with a Teflon tap. It was partially filled to a height of200 mm with a weak anion exchange resin (Diaion WA-30 resin averageparticle size 0.47 mm, total exchange capacity 1.5 meq/mL suppied bySupelco). The resin bed volume was 50 mL.

Column D: The column was glass with internal diameter 23 mm and height180 mm fitted with a Teflon tap. It was partially filled to a height of180 mm with resin (Amberlite XAD-16 Surface Area 800 m2/g, Average PoreDiameter 100 Angstroms supplied by Sigma-Aldrich). The resin bed volumewas 50 mL.

The OPE and polymer adsorbent resins were used as supplied or wereprepared prior to use in accordance with the general proceduresdescribed above.

The elution of bioactive compounds from each column was monitored byHigh Performance Liquid Chromatography (Shimadzu VP7 HPLC systemconsisting of low-pressure mixing system FCV-10AL, degassing systemDGU-14A, solvent delivery module LC-10AD, autosampler SIL-10AD, diodearray detector SPD-M10A VP and VP software.)

Column a (Acrylic Polymer Adsorbent):

The acrylic polymer adsorbent was shown in this experiment to separatelimonoid glucosides from flavonoid compounds.

Six litres of 4.1 Brix OPE were loaded onto the column containing theacrylic polymer adsorbent (column A) in 1 litre aliquots. The OPE wasallowed to percolate through the column after the application of eachaliquot.

Initially, after 1 litre of the OPE had passed through the column, nolimonoid or flavonoid compounds are eluted from the column. After 2litres of OPE had been applied to the column, limonoid compounds(principally limonin glucoside at retention time 19 minutes) wereobserved to elute from the column. After a total of 3, 4 and 5 litres ofOPE had been applied to the acrylic resin, more limonoid compounds,principally limonin glucoside (LG), deacetyl nomilin glucoside (DANG)(retention time 24.5 minutes), nomilin glucoside (NG) (retention time28.4 minutes) and nomilic acid glucoside (NAG) (retention time 29minutes) were observed to be eluted from the acrylic resin. After 6litres of OPE had been applied to the column, obacunone glucoside (OG)(retention time 31 minutes) was observed to be eluted from the column inaddition to the limonoid compounds identified previously.

A HPLC chromatogram of the eluate obtained after 6 litres of OPE haseluted through Column (A) is shown in FIG. 9. The relative amounts ofeach limonoid glucoside compound eluted from the acrylic polymer resinmay be determined and a graph illustrating the relative amounts of eachlimonoid glucoside compound is shown in FIG. 10.

In none of the collected eluates was the presence of any of the majorflavonoid compounds detected. Consequently, it is shown that the acrylicpolymer adsorbent was able to substantially remove the flavonoidcompounds from the OPE while the limonoid compounds, which were notadsorbed on to the acrylic resin, were allowed to pass through.

As discussed below, the OPE fractions eluted from Column (A) may then beloaded onto Column (B) to purify the limonoid glucosides and to alsoprepare a palatable “juice” from the natural sugars and other highlypolar compounds present in the OPE.

Once the OPE had been allowed to pass through the acrylic resin, thecolumn was then washed with two bed volumes of water. Further quantitiesof limonoid compounds were eluted from the column with the water howeverno flavonoid compounds were observed to be present in the water eluate.

An aqueous ethanol eluent solution was then applied to the top of thecolumn to elute the flavonoid compounds that had been adsorbed on to theacrylic polymer resin. A gradient alcohol concentration that increasedin a step-wise manner from 20% (v/v) to 60% (v/v) was applied inaccordance with Table 7. After application, each aliquot of eluent wasallowed to percolate through the column to desorb the bioactiveflavonoid compounds from the acrylic resin. The desorbed compounds weresubsequently collected and analysed by HPLC.

TABLE 7 Fraction Strength (% Ethanol) Bed Volumes 15 20 1 16 30 1 17 401 18 50 1 19 60 1 20 80 1

The eluate obtained after desorbing the acrylic resin (column (A)) witha solution containing 20% ethanol did not contain any flavonoidcompounds. However, when the concentration of ethanol in the eluent wasincreased to 30%, the resulting eluate did contain a small amount offlavonoids. Further increasing the ethanol concentration in the eluateto 40% and 50% resulted in significant amounts of flavonoid compoundsnarirutin (retention time 22 minutes) and hesperidin (retention time24.35 minutes) being desorbed and eluted from the acrylic resin. Whenthe amount of ethanol in the eluent was increased to 60%, the resultingeluate contained a significant amount of narirutin and hesperidin aswell as didymin at a retention time of 27 minutes.

A HPLC chromatogram of an eluate fraction obtained after desorption ofthe flavonoid compounds from column (A) with 40% ethanol is shown inFIG. 11. The relative amounts of each flavonoid compound desorbed fromthe acrylic polymer resin by each eluent fraction may be determined anda graph illustrating the relative quantities of flavonoid compoundscollected as the alcohol concentration increased is shown in FIG. 12.

It has been found that the acrylic polymer resin holds up theflavonoids, but allows the limonoid glucosides to pass through alongwith more polar polyphenolics, sugars and organic acids. The flavonoidscould then be desorbed from the acrylic resin using aqueous ethanol.

Column B (Polystyrene-Divinyl Benzene Adsorbent):

The polystyrene-divinylbenzene column is shown in the experiment to beuseful in the separation of the limonoid glucosides from the naturalsugars and more polar compounds eluting from Column (A).

In this trial, a total of six litres of 4.1 Brix OPE from Column (A)were loaded onto Column (B) containing the polystyrene-divinyl benzenepolymer absorbent, in 1 L aliquots as they came off the acrylic resincolumn. The OPE aliquots were allowed to percolate through thepolystyrene-divinyl benzene resin and the eluted aliquots were collectedand analysed by HPLC.

After application of the first two aliquots of OPE onto Column (B), nolimonoid glucosides were observed to be eluted from the column. Afterapplication and elution of the third and fourth aliquots of OPE, a smallamount of limonoid glucosides (but no flavonoid compounds) was observedto pass through the column. After application of the fifth and sixthaliquots of OPE on to Column (B), some limonin glucoside and smallamounts of other limonid glucosides eluted from the column. The columnwas then washed with four bed volumes of water to remove the naturalsugars and other highly polar material from the column. The waterfractions were collected and may be combined with the eluted OPEfractions to form a palatable “juice” component that is free of anybitter compounds A HPLC chromatogram of the “juice” component obtainedfrom column (B) is shown in FIG. 13. The OPE eluates obtained fromColumn (B) did not have the major flavonoids (principally hesperidin,narirutin and didymin), nor did they contain the five limonoidglucosides, limonin glucoside, deacetyl nomilin glucoside, nomilinglucoside, nomilic acid glucoside and obacunone glucoside. The OPEeluate obtained from Column (B) contained polyphenolics plus sugars andorganic acids. The eluate is suitable for blending back into foodproducts such as orange juice, to supplement the food product.

The limonoid glucosides retained on Column (B) were then desorbed fromthe column with an aqueous ethanol eluent solution. As shown in Table 8,a stepped ethanol gradient that increased from 10% (v/v) to 80% (v/v)was used as the eluent.

TABLE 8 Fraction Strength (% Ethanol) Bed Volumes 21 10 2 22 20 2 23 302 24 50 2 25 80 2

After application, each aliquot of eluent was allowed to percolatethrough the column to desorb the limonoid glucoside compounds fromColumn (B) and were subsequently collected and analysed by HPLC.

It was observed that the eluate obtained after desorbing thepolystyrene-divinyl benzene polymer resin (column B) with a solutioncontaining 10% ethanol contained some limonoid glucosides, principallydeacetyl nomilin glucoside (retention time 25 minutes) and nomilinglucoside (retention time 29 minutes).

Upon increasing the concentration of alcohol in the eluent to 20%, otherlimonoid glucoside compounds, limonin glucoside, nomilinic acidglucoside and obacunone glucoside were observed to elute from Column(B). Further increases in he concentration of ethanol in the eluent to30%, 50% and 80% resulted in greater quantities of the five limonoidglucosides being desorbed from the polystyrene-divinyl benzene polymerresin.

A HPLC chromatogram of an eluate fraction obtained after desorption ofthe limonoid glucoside compounds from column (B) is shown in FIG. 14.The relative amounts of each limonoid glucoside compound desorbed fromthe polystyrene-divinyl benzene polymer resin in each eluate fractionmay be determined and a graph illustrating the relative quantities ofeach limonoid glucoside collected is shown in FIG. 15.

Column C (Weak Anion Exchange Resin):

The weak anion exchange column is shown in this experiment to be usefulin the separation of the limonoid glucosides from the more neutralcompounds eluting from Column (B).

The eluates containing the limonoid glucosides desorbed from Column (B)were combined and diluted with water to a strength of around 20% ethanolby volume. The combined eluates were then applied to the top of Column(C) and allowed to percolate through the column at a rate of about tenbed volumes per hour. The liquid passing through the column wasdiscarded. However, it is envisaged that in a commercial application,the liquid could be diverted and the alcohol subsequently recovered.HPLC analysis showed that the liquid which passed through column (C) didnot contain any limonoid glucosides.

The limonoid glucosides were then desorbed from column (C) using asolution of 0.5M sodium chloride (brine) solution. Almost 100% of thelimonoid gluco sides had eluted within eight bed volumes of brinepassing through the column. A HPLC chromatogram of the eluate obtainedafter passing a brine solution through the anion exchange resin ofcolumn (C) is shown in FIG. 16. A graph illustrating the amount of eachlimonoid glucoside desorbed from the anion exchange resin as increasingquantities of brine is applied to the resin is shown in FIG. 17.

Column D (Polystyrene-Divinyl Benzene Adsorbent):

The polystyrene-divinylbenzene polymer adsorbent was shown in thisexperiment to be useful in removing the sodium chloride salt from thelimonoid glucoside compounds eluting from Column (C). The salt wasobserved to pass through the polystyrene-divinyl benzene polymeradsorbent of column (D) while the limonoid glucosides were retained bythe polymer.

The brine solution containing the desorbed limonoid glucoside compoundsobtained from column (C) is applied to the top of thepolystyrene-divinyl benzene resin of column (D) and allowed to percolatethrough the polymer resin. The limonoid glucosides were observed toadsorb on to the polymer adsorbent while the sodium chloride in thebrine does not. Column (D) was then washed with four bed volumes ofwater to remove the sodium chloride salt.

The limonoid glucosides were then desorbed from the polystyrene-divinylbenzene polymer resin with an eluent solution containing 50% aqueousethanol. The HPLC chromatogram of an eluate fraction collected afterdesorption of the limonoid glucosides is shown in FIG. 18.

An outcome of the process is that a concentrated limonoid glucosidefraction can be obtained. As seen in stacked HPLC chromatograms of FIG.19 showing the original Orange Peel Extract (lower line in eachchromatogram) and the concentrated limonoid glucoside fraction (upperline in each chromatogram), the concentration of LG's in the originalOPE are about 1000 parts per million in total, and individual peaks arehard to see. In the resultant concentrated fraction however, the LG'samount to more than 80% of the components in the concentrate. Given that12 Brix OPE contains 1000 ppm LG's in toto, every litre contains 100 mgof pure LG's. Hence the process can produce one gram of LG's per litreof acrylic resin for each cycle of the process.

The above example demonstrates that different polymer adsorbent resinscan be used to separate and purify bioactive compounds from orange peelextract without significant losses of the target compounds.

It is understood that various other modifications and/or alterations maybe made without departing from the spirit of the present invention asoutlined herein.

1. A process for the separation of bioactive compounds, the processcomprising the steps of: (a) contacting a plurality of bioactivecompounds with a first polymer adsorbent under conditions allowingadsorption of at least one bioactive compound on to the first adsorbentwhile at least one bioactive compound is not adsorbed on to the firstadsorbent; (b) collecting a solution comprising the at least onebioactive compound which has not adsorbed on to the first adsorbent; and(c) contacting the solution obtained in step (b) with a second polymeradsorbent under conditions allowing adsorption of at least one bioactivecompound contained in the solution obtained in step (b) on to the secondadsorbent, wherein the first polymer adsorbent and the second polymeradsorbent comprise different polymeric materials.
 2. A process accordingto claim 1, wherein the plurality of bioactive compounds comprises aflavanone glycoside and a limonoid glucoside, and wherein the flavanoneglycoside is adsorbed on to the first polymer adsorbent and the limonoidglucoside is adsorbed on to the second polymer adsorbent tosubstantially separate the flavanone glycoside and the limonoidglucoside.
 3. A process according to claim 1, wherein the first polymeradsorbent is an acrylic.
 4. A process according to claim 3, wherein thefirst polymer adsorbent is an acrylic ester.
 5. A process according toclaim 4, wherein the first polymer adsorbent is polymethylmethacrylate.6. A process according to claim 1, wherein the second polymer adsorbentis capable of adsorbing to non-polar compounds.
 7. A process accordingto claim 1, wherein the second polymer adsorbent is polystyrene-divinylbenzene.
 8. A process according to claim 1, wherein the first polymeradsorbent and the second polymer adsorbent are each arranged in acolumn.
 9. A process according to claim 1, further comprising the stepsof: contacting the at least one bioactive compound adsorbed on the firstpolymer adsorbent with an eluent under conditions allowing desorption ofthe at least one bioactive compound from the first adsorbent; andeluting the at least one bioactive compound from the first absorbent.10. A process according to claim 9, wherein the eluent comprises alcoholand water.
 11. A process according to claim 10, wherein theconcentration of alcohol in the eluent remains substantially constantduring desorption of the at least one bioactive compound from the firstpolymer adsorbent.
 12. A process according to claim 10, wherein theconcentration of alcohol in the eluent increases during desorption ofthe at least one bioactive compound from the first polymer adsorbent.13. A process according to claim 1, further comprising the steps of:contacting the at least one bioactive compound adsorbed on the secondpolymer adsorbent with an eluent under conditions allowing desorption ofthe at least one bioactive compound from the second adsorbent; andeluting the at least one bioactive compound from the second absorbent.14. A process according to claim 13, wherein the eluent comprisesalcohol and water.
 15. A process according to claim 14, wherein theconcentration of alcohol in the eluent remains substantially constantduring desorption of the at least one bioactive compound from the secondpolymer adsorbent.
 16. A process according to claim 14, wherein theconcentration of alcohol in the eluent increases during desorption ofthe at least one bioactive compound from the second polymer adsorbent.17. A process according to claim 13, further comprising the step of:contacting the at least one bioactive compound eluted from the secondpolymer adsorbent with an ion exchange resin under conditions allowingionic interactions between the at least one bioactive compound and theresin such that the at least one bioactive compound is adsorbed on tothe resin.
 18. A process according to claim 17, wherein the ion exchangeresin is an anion exchange resin.
 19. A process according to claim 18,wherein the ion exchange resin is a weak base anion exchange resin. 20.A process according to claim 17, wherein the ion exchange resin isarranged in a column.
 21. A process according to claim 17, furthercomprising the steps of: contacting the at least one bioactive compoundadsorbed on the ion exchange resin with a solution comprising a soluteunder conditions allowing the solute to displace the at least onebioactive compound from the resin; and eluting the at least onebioactive compound from the resin.
 22. A process according to claim 21,wherein the solute is a salt.
 23. A process according to claim 22,wherein the salt is sodium chloride.
 24. A process according to claim 1,wherein the plurality of bioactive compounds are obtained from a citrusfruit.